Regulation of Lipid Metabolism and Membrane Trafficking by the Oxysterol Binding Protein Superfamily Member Kes1
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The Saccharomyces cerevisiae oxysterol binding protein homologue Kes1/Osh4 is a member of an enigmatic class of proteins found throughout Eukarya. This family of proteins is united by a ?-barrel structure that binds sterols and oxysterols. An N-terminal lid is thought to both sequester sterols inside the core and promote localization of Kes1 to regions of high membrane curvature via a predicted ArfGAP lipid packing sensor motif. Additionally, a phosphoinositide-binding region on a discrete surface of Kes1 has also been identified. In this thesis, structure-function analysis of Kes1 determined that phosphoinositide binding is required for membrane association in vitro, and in vivo phosphoinositide binding is required for localization to the Golgi. Ergosterol, the major sterol in S. cerevisiae, and membrane curvature had minimal effects on membrane association. This study also revealed a role for Kes1 in the regulation of both phosphatidylinositol-4-phosphate and phosphatidylinositol-3-phosphate homeostasis. Phosphoinositide and sterol binding by Kes1 are necessary for it to alter phosphatidylinositol-4-phosphate, but not phosphatidylinositol-3-phosphate homeostasis. Misregulation of phosphatidylinositol-4-phosphate homeostasis by Kes1 manifested itself in an inability of the v-SNARE Snc1 to traffic properly and was consistent with a defect in trans-Golgi/endosome trafficking. I went on to demonstrate a role for Kes1 in regulating the conversion of phosphatidylinositol-4-phosphate to phosphatidylinositol for the synthesis of sphingolipids, and I present a model for the role of Kes1 at the Golgi. Kes1 acts as a sterol sensor that regulates phosphatidylinositol-4-phosphate to sphingolipids metabolism, which ultimately regulates the delivery of proteins that assemble into lipid rafts for their transport from the Golgi to the plasma membrane. I also uncovered a previously unknown role for Kes1 in the regulation of the cytoplasm-to-vacuole and autophagy trafficking pathways, which is mediated by the ability of Kes1 to regulate phosphatidylinositol-3-phosphate homeostasis.